Catalytic glycosylation with designer thioglycoside and novel protecting groups for same and for synthesis of oligosaccharides

ABSTRACT

A catalytic glycosylation method comprising: installing thioether to an anomeric carbon of a carbohydrate; and catalytically activating the thioether with a non-oxophilic Lewis acid. The thioether may comprise an anomerically stable thioether leaving group. The catalytic glycosylation method may further comprise: utilizing an acid-sensitive ester protecting group as permanent protecting group or using a reactivity-based one-pot glycosylation that employs a single-component catalyst to accelerate an oligosaccharide assembly process. A protecting group to mask hydroxyl functionalities in the production of oligosaccharides, natural products or any molecule having a hydroxyl group comprising an acid-labile ester protecting group.

RELATED APPLICATIONS

This application claims priority benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/656,366, filed Jun. 6, 2012, and of U.S.Provisional Application No. 61/677,993, filed Jul. 31, 2012 the contentsof which are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure generally relates to catalytic glycosylationmethods and protecting groups for the same and for synthesis ofoligosaccharides, natural products or any molecule having a hydroxylgroup.

BACKGROUND

Oligosaccharide is the third most abundant biopolymer in a livingsystem, next to nucleic acid and proteins. The biological significanceof oligosaccharide is undisputable, yet the rapid preparation ofhomogeneous oligosaccharide by automation, analogues to the synthesis ofDNA/RNA oligonucleotides and peptides, remains far beyond reach.

Two of the most fundamental issues in modern chemical synthesis ofoligosaccharides that requires innovation are 1) chemical glycosylationmethod that permits the robust construction of desired glycosidiclinkage, 2) protecting groups that can be strategically applied to theblockage of designated hydroxyl, amino, carboxyl groups, yet can bereadily removed to release the desired oligosaccharide. The presentdisclosure addresses both of these fundamental issues with respect tomodern chemical synthesis of oligosaccharides.

FIG. 1 shows a comparison of known catalytic glycosylation methods witha preferred catalytic glycosylation method of the present disclosure.Currently available chemical glycosylating agents largely fall into twocategories. One type is based on anomerically labile leaving groups,which can be activated by catalytic amount of a Lewis acid. Theclassical example is trichloroacetaimidate based glycosylating agent(Schmidt donor), but also includes glycosyl phosphite and ester-basedglycosylating agent. These glycosylating agents do not tolerateacid/base treatment so that the leaving group itself has to be installedin the last step of the monosaccharide or oligosaccharide building blockpreparation prior to the actual glycosylation event as shown in FIG. 1.From a practical point-of-view, this is a critical drawback, as thepreparation of any imidate-type glycosylating agent requires thepre-selection of a protecting group to mask the anomeric center andremove it at the penultimate step to install the leaving group.

The other type of widely used glycosylating agent is based onanomerically stable leaving group. The classical examples are thioetheror n-pentenyl ether based glycosylating agents. While these types ofleaving groups are anomerically stable, they have to be activated bymore than stoichiometric amount of activator and require the usage ofextra component, such as bulky non-nucleophilic amine base toeffectively quench the in-situ generated acid.

Therefore, one would envision that an ideal type of chemicalglycosylating agent should combine the catalytic activator-feature ofglycosyl imidate and the anomeric stable feature of thioglycoside.Preferred glycosylation methods of the present disclosure fulfill thiscriterion. Moreover, the most commonly used activators in chemicalglycosylation are highly oxophilic Lewis acids or thiophilicelectrophiles. In both cases, the reaction will be carried out in anacidic environment, which not only calls for the extra non-nucleophilicbase (not atom-economical) but also preludes the application ofacid-sensitive protecting groups as permanent protecting groups inoligosaccharide assembly. The preferred glycosylation methods of thepresent disclosure provide a new class of thioglycoside which permitsthe application of cationic gold(I) complex as an activator, which iscarbophilic rather than oxophilic, thus circumventing the limitationassociated with the usage of oxophilic Lewis acid with conventionalglycosylation agents.

Another fundamental issue in modern chemical synthesis ofoligosaccharides is that too many orthogonal protecting groups forhydroxyl and amino functionalities are introduced at the early stage ofthe process. While the adoption of this strategy is clearlyunderstandable, as the carbohydrate backbone contains a myriad ofhydroxyls and amines which have to be “chemically protected” properly inorder to achieve regioselective chain elongation, the excessorthogonalities in terms of chemical reactivity that are present in aprotected oligosaccharide make the late stage chemical synthesis tediouswhich often results in unpredictable failure.

Benzyl ethers and ester-type of protecting groups are two most commonlyused hydroxyl protecting groups in carbohydrate synthesis that requiresdifferent chemical treatment for removal. While benzyl ethers areusually sensitive to hydrogenolysis and acid, esters are sensitive tobase-catalyzed hydrolysis. Within the present disclosure, it isdesirable to design a series of hydroxyl protecting groups that retainthe basic properties of benzyl ethers and esters, but can be deprotectedby a common type of chemical reagent, acid. This aspect of the presentdisclosure will dramatically speed up the chemical synthesis ofoligosaccharide, particularly allowing for the automation process, whencoupled with a glycosylating agent that does not require strong acid foractivation.

SUMMARY

One aspect of a preferred embodiment of the present disclosure comprisesa catalytic glycosylation method comprising: installing thioether to ananomeric carbon of a carbohydrate; and catalytically activating thethioether with a non-oxophilic Lewis acid.

In another aspect of a preferred catalytic glycosylation method of thepresent disclosure, the thioether comprises an anomerically stablethioether leaving group.

In a further aspect, a preferred catalytic glycosylation method furthercomprises utilizing an acid-sensitive ester protecting group aspermanent protecting group.

In yet another aspect, a preferred catalytic glycosylation methodfurther comprises using a reactivity-based one-pot glycosylation thatemploys a single-component catalyst to accelerate an oligosaccharideassembly process.

In a further aspect, a preferred catalytic glycosylation method furthercomprises utilizing an application of a 100%-PEG-based polymer asinsoluble support for solid-phase oligosaccharide synthesis.

In yet an additional aspect, a preferred catalytic glycosylation methodfurther comprises utilizing a designer thioglycoside that retains basicproperties of a parental thioglycoside, including the ease ofpreparation and toleration of backbone protecting group manipulation.

In yet another aspect, a preferred catalytic glycosylation methodfurther comprises applying an activator permitting an application ofhighly acid-sensitive protecting groups; applying a 100%-PEG-basedpolymer as insoluble support for solid-phase oligosaccharide synthesisto streamline an oligosaccharide assembly.

In another aspect of a preferred catalytic glycosylation method of thepresent disclosure the activator is carbophilic.

In another aspect of a preferred catalytic glycosylation method of thepresent disclosure the activator is a cationic gold(I) complex.

Another aspect of a preferred embodiment of the present disclosurecomprises a method of synthesizing an oligosaccharide, comprising thesteps of: tethering an acetyl ester and a benzoyl ester to a saccharidewith an alcohol group; and protecting the alcohol group with anacid-labile ester protecting group.

In a further aspect, a preferred method of synthesizing anoligosaccharide further comprises the step of deprotecting the estergroup by acid treatment.

An additional aspect of a preferred embodiment of the present disclosurecomprises a method of synthesizing an oligosaccharide comprising thestep of activating a thioglycoside with a non-oxophilic Lewis acid.

In another aspect of a preferred method of synthesizing anoligosaccharide of the present disclosure the Lewis acid comprises acationic gold(I) complex.

A further aspect of a preferred embodiment of the present disclosurecomprises a method of synthesizing an oligosaccharide, comprising thesteps of: tethering an acetyl ester and a benzoyl ester to athioglycoside with an alcohol group; protecting the alcohol group withan acid-labile ester protecting group; deprotecting the ester group byacid treatment; and activating the thioglycoside with a non-oxophilicLewis acid.

In another aspect of a preferred method of synthesizing anoligosaccharide of the present disclosure the Lewis acid comprises acationic gold(I) complex.

Another aspect of a preferred embodiment of the present disclosurecomprises a protecting group to mask hydroxyl functionalities in theproduction of oligosaccharides, natural products or any molecule havinga hydroxyl group comprising an acid-labile ester protecting group.

In another aspect of a preferred protecting group of the presentdisclosure, the acid-labile ester protecting group is selected from agroup consisting of an acetyl ester tethered with a para methoxybenzyl(PMB) ether, an acetyl ester tethered with a napthyl methyl (NAP) ether,a benzoyl ester tethered with a PMB ether and a benzoyl ester tetheredwith an NAP ether.

In a further aspect of a preferred protecting group of the presentdisclosure, the tethering of an acetyl ester or a benzoyl ester with analcohol group that is protected with an acid-labile ester protectinggroup can be de-protected by an acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is illustrated by way of example and notlimitation in the figures of the accompanying drawings, in which:

FIG. 1 shows a comparison of known catalytic glycosylation methods (top)with a preferred catalytic glycosylation method of the presentdisclosure (bottom).

FIG. 2 shows a summary of the novel features of the glycosylatingmethods/systems according to preferred embodiments of the presentdisclosure.

FIG. 3 illustrates a first preferred catalytic glycosylation method ofthe present disclosure.

FIG. 4 shows another preferred catalytic glycosylation method of thepresent disclosure.

FIG. 5 illustrates an additional preferred catalytic glycosylationmethod of the present disclosure.

FIG. 6 shows yet another preferred catalytic glycosylation method of thepresent disclosure.

FIG. 7 illustrates reactivity-based glycosylation according to preferredembodiments of catalytic glycosylation methods of the presentdisclosure.

FIG. 8 illustrates additional reactivity-based glycosylation accordingto preferred embodiments of catalytic glycosylation methods of thepresent disclosure.

FIG. 9 shows preferred processes for attaching the designed thioethersto carbohydrates and transforming to the designed glycosylating agentswith respect to preferred catalytic glycosylation methods of the presentdisclosure.

FIG. 10 illustrates the compatibility of preferred glycosylation agentsto known protecting group manipulations for use in preferred catalyticglycosylation methods of the present disclosure.

FIG. 11 illustrates differences between known protecting groups (top)and preferred acid-labile ester protecting groups for use in preferredmethods of the present disclosure.

FIG. 12 illustrates preferred examples of acid-sensitive groupsaccording to preferred embodiments of using novel protecting groups ofthe present disclosure.

FIG. 13 illustrates preferred acidic conditions for removing acid-labileester protecting groups according to preferred embodiments of thepresent disclosure.

FIG. 14 shows that preferred acid-labile ester protecting groups of thepresent disclosure are chemically compatible as substitutes for otherknown protecting groups.

FIG. 15 illustrates shows that preferred acid-labile ester protectinggroups of the present disclosure may be employed as a global protectinggroup protecting all hydroxyl groups on an oligosaccharide backboneaccording to preferred embodiments of the present disclosure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following description, taken in conjunction with the referenceddrawings, is presented to enable one of ordinary skill in the art tomake and use the disclosure and to incorporate it in the context ofparticular applications. Various modifications, as well as a variety ofuses in different applications, will be readily apparent to thoseskilled in the art, and the general principles, defined herein, may beapplied to a wide range of aspects. The present disclosure is notintended to be limited to the aspects disclosed herein. Instead, it isto be afforded the widest scope consistent with the disclosed aspects.

In essence, the present disclosure details the rational design ofpreferred anomerically stable thioglycosides that can be catalyticallyactivated by cationic gold (I) complex. The glycosylating methods/systemaccording to preferred embodiments of the present disclosure are novel,as they represent the first disclosed glycosylation platform whichfeatures an anomerically stable leaving group that can be activated by acatalytic amount of a single component activator. The activator itself(cationic gold(I) complex) is a non-oxophilic Lewis acid that permitsthe application of highly acid-sensitive protecting groups, as describedherein, as global protecting groups to dramatically streamline thecomplex oligosaccharide synthesis. The overall system is both robust andmodular in terms of the glycosylating agent itself and the activator,the reactivity of which can be readily tuned to streamline theoligosaccharide assembly process.

FIG. 2 shows a summary of the novel features of the glycosylatingmethod/system according to preferred embodiments of the presentdisclosure including:

A preferred and the first catalytic glycosylation system that featuresan anomerically stable thioether leaving group.

The preferred catalytic glycosylation methods/systems permit theapplication of highly acid-sensitive protecting groups as permanentprotecting group using a series of preferred acid-sensitive ester typeprotecting groups described herein.

The preferred catalytic glycosylation methods/systems permit thereactivity-based one-pot glycosylation that employs a single-componentcatalyst that dramatically accelerates the oligosaccharide assemblyprocess.

The preferred catalytic glycosylation methods/systems permit theapplication of 100%-PEG-based polymer as insoluble support forsolid-phase oligosaccharide synthesis which cannot be achieved withtraditional oxophilic Lewis acid activator, as they will bind the PEGbackbone and diminish their activities as activators.

The designer thioglycoside according to preferred embodiments of thepresent disclosure retains the basic properties of parentalthioglycoside, including the ease of preparation and toleration ofbackbone protecting group manipulation, an essential feature forpreparative purpose.

FIGS. 3-5 illustrate first preferred catalytic glycosylation methods ofthe present disclosure representing the first catalytic glycosylationmethods featuring an anomerically stable thioether leaving group. Thepreferred catalytic glycosylation methods are modular both in terms ofthe glycosylating agent, where the backbone of thioaryl ether can bereadily modified to change its reactivity and also the activator. Thepreferred catalytic glycosylation methods only require a singlecomponent cationic gold(I) complex as the activator, which isdrastically different from conventional chemistry involvingthioglycoside activation. The by-product generated in the preferredcatalytic glycosylation methods of the present disclosure(benzothiophene) does not participate the glycosylation, which isdifferent from known glycosyltrichloroimidate chemistry where theby-product trichloroacetamide can serve as competitive nucleophile tocomplicate the glycosylation reaction.

As shown in FIG. 6, the preferred catalytic glycosylation methods of thepresent disclosure permit the application of highly acid-sensitiveprotecting groups, described herein, as permanent protecting group.These types of -transformations cannot be routinely carried out withglycosyl imidates or conventional thiolgycoside.

FIGS. 7-8 illustrate additional preferred catalytic glycosylationmethods of the present disclosure which permit the reactivity-basedone-pot glycosylation that employs a single-component catalyst thatdramatically accelerates the oligosaccharide assembly process. FIG. 7shows reactivity based catalytic glycosylation while FIG. 8 illustratesreactivity based catalytic glycosylation to access blood antigenoligosaccharide.

The preferred catalytic glycosylation methods of the present disclosurewhich permit the application of 100%-PEG-based polymer as insolublesupport for solid-phase oligosaccharide synthesis. This cannot beachieved with traditional oxophilic Lewis acid activator, as they willbind the PEG backbone and diminish their activities as activators.100%-PEG-based polymer is marketed by Novabiochem and has been widelyapplied in peptide synthesis. The preferred designer thioglycosides ofthe present disclosure retain the basic properties of parentalthioglycoside, including the ease of preparation and toleration ofbackbone protecting group manipulation, an essential feature forpreparative purpose.

Novel Protecting Groups for Synthesis of Oligosaccharides and NaturalProducts

The present disclosure preferably employs a series of ester-type ofprotecting groups that are used to mask hydroxyl functionalities. Whiletraditional ester protecting groups require base treatment for removal,by tethering acetyl ester and benzoyl ester with an alcohol group thatis protected with an acid-labile protecting group, the ester group canbe readily deprotected by acid treatment. The preparation of thisester-protecting group is straightforward and it can be done on amulti-gram scale in a routine academic lab. By tuning the ester backboneas well as the tethered alcohol protecting group, a set of newacid-responsive ester protecting groups is preferably obtained. This notonly can be used as temporary protecting group from complex carbohydrateand natural product synthesis, but can also be used as permanentprotecting group for complex carbohydrate synthesis, as outlined in FIG.15 showing the synthesis of an oligomannoside according to a preferredembodiment of the present disclosure.

FIG. 9 shows preferred processes for attaching the designed thioethersto carbohydrates and transforming to the designed glycosylating agentswith respect to preferred catalytic glycosylation methods of the presentdisclosure. FIG. 10 illustrates the compatibility of preferredglycosylation agents to known protecting group manipulations for use inpreferred catalytic glycosylation methods of the present disclosure.

The following examples/schemes, as depicted in FIGS. 11-15, illustratepreferred aspects of oligosaccharide synthesis using novel protectinggroups of the present disclosure. The preferred embodiments of thepresent disclosure will streamline the synthesis of biologicallyimportant oligosaccharide by automation. To the best of the inventor'sknowledge, no acid sensitive ester-type protecting group has ever beendescribed in the context of complex molecule synthesis. The presentdisclosure allows for the dramatic enhancement of production efficiencyof biologically active compounds in both industrial and academic labswhich are oriented towards biological research.

It should be emphasized the technical difficulties associated with thepreparation of oligosaccharides largely exceeds those of DNA, RNA andpeptides. RNA, a homologue of DNA, but with an extra hydroxyl group atC-2 position of ribose, was once considered difficult to synthesize byautomation, because of the lack of proper protecting group to mask thatfunctionality.

It should be understood that while this disclosure has been describedherein in terms of specific, preferred embodiments set forth in detail,such embodiments are presented by way of illustration of the generalprinciples of the disclosure, and the disclosure is not necessarilylimited thereto. Certain modifications and variations in any givenmaterial, process step or chemical formula will be readily apparent tothose skilled in the art without departing from the true spirit andscope of the present disclosure, and all such modifications andvariations should be considered within the scope of the claims thatfollow.

What is claimed is:
 1. A catalytic glycosylation method comprising:installing an anomerically-stable thioether leaving group onto ananomeric carbon of a carbohydrate to yield an ortho-alkynylphenylthioglycoside, wherein the anomerically stable thioether leaving groupis ortho-alkynylthiophenol; and catalytically activating theanomerically-stable thioether leaving group with an activator comprisingPPh₃AuNTf₂.
 2. The catalytic glycosylation method of claim 1, whereinthe thioglycoside has hydroxyl functionality, the method furthercomprising: a step of masking the hydroxyl functionality with apermanent protecting group, the permanent protecting group selected fromthe group consisting of

wherein R is a para-methoxybenzyl or napthyl methyl moiety.
 3. Thecatalytic glycosylation method of claim 1, further comprising: using theortho-alkynylphenyl thioglycoside in a reactivity-based one-potglycosylation that employs the activator as a single-component catalystto accelerate an oligosaccharide assembly process.
 4. The catalyticglycosylation method of claim 1, further comprising: using theortho-alkynylphenyl thioglycoside in a solid-phase oligosaccharidesynthesis employing a 100%-PEG-based polymer as insoluble support forthe solid-phase oligosaccharide synthesis.